X4S-6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



active culture grown on agar are shaken into a water suspension and 

 are commonly spread on the seed just before planting. Many com- 

 mercial "inoculations" are prepared by mixing the culture with some 

 moisture absorbing powder, such as autoclaved ground peat. Wood 

 flour is also quite absorbent, and contains almost no bacteria. If the 

 seed is drill-sown, it is made only moist enough to distribute the bac- 

 teria well, and then dried sufficiently not to clog the drill. To secure 

 uniform results it is best to use plenty of bacterial culture, for 

 example, 5000 bacteria (plate count) per seed. For convenience 

 in estimating the number of bacteria per seed a brief table is given 

 by Fred, Baldwin and McCoy (1932), w^ho review this general 

 subject, showing the average number of seeds per pound of many 

 legumes. 



SPRAY INOCULATION 



Spraying is one of the methods most commonly used in plant inoc- 

 ulation. It is particularly useful in diseases where the bacteria enter 

 the host plants through natural openings such as stomata, water 

 pores, and nectaries. For many simple tests, suspensions of bacteria 

 are merely sprayed on the surfaces of susceptible leaves, stems, flow- 

 ers, fruits, etc. For more exact tests, however, such as those for com- 

 parative virulence, it is common to suspend the growth from an agar 

 culture in water, saline solution (0.9% NaCl), or a selected buffer 

 (such as suitable mixtures of dilute K2HPO4 and KH2PO4), and to 

 standardize the concentration according to a selected and measured 

 turbidity. If the bacteria have been grown in liquid culture, the 

 entire culture may be used. This procedure, however, is often un- 

 satisfactory because, after spraying, secondary organisms may grow 

 in the nutrient medium. It is frequently better to separate the 

 bacteria from the medium by means of a centrifuge and to resuspend 

 the cells as with the growth from agar media. 



The number of bacteria in a suspension may be determined, for 

 example, (1) by direct examination in a Petroff-Hausser counting 

 chamber; or (2) by mixing a known volume of the bacteria with previ- 

 ously counted suspensions of yeast or red blood cells, making smears, 

 and determining the relative number of bacteria and cells. Bacterial 

 suspensions are often duplicated by comparing their turbidity with 

 that of a graded series of barium sulfate standards (described by Riker 

 and Riker, 1936). A common density for a bacterial suspension has 

 the turbidity of a solution obtained by mixing 1 ml. of a 1% solution 

 of barium chloride with 99 ml. of dilute sulfuric acid. Turbidity can 

 be measured accurately and rapidly in an Evelyn densiometer. 



The prepared bacterial suspension is filtered through cheesecloth, 

 to remove small pieces of agar or other materials which might clog 

 the spray nozzle, and is placed in the spraying device. The plants are 

 sprayed so that good coverage is given especially to the lower sides of 

 leaves which commonly have more stomata. The plants are placed in 

 an environment where they will not dry off for a number of hours. 



Certain additional precautions are sometimes necessary for best 

 results, of which several are mentioned briefly. (1) The relative 

 humidity of the air surrounding the host plant is maintained at satu- 



